The present invention relates to novel benzhydrylamine derivatives that are useful as reaction reagents for the solid-phase synthesis of polypeptide amides making use of a 9-fluorenylmethyloxycarbonyl group (hereinafter abbreviated as "Fmoc"). The present invention also relates to an intermediate of such derivatives.
The use of Fmoc, which is the base-labile protecting group developed by Carpino and Han, in the automated solid-phase synthesis of polypeptides has been known in the art (Carptino, L. A., Han, G. Y.: J. Am. Chem. Soc., 92, 5748 (1970); Carpino, L. A.: Acc. Chem. Res., 20, 401 (1987)). This method employs, in combination with Fmoc, a t-butoxycarbonyl (hereinafter abbreviated as "Boc") or t-butyl (hereinafter abbreviated as "tBu") ester or ether as an acid-labile group for protecting side chains in amino acids. Protecting groups such as Boc and tBu can be cleaved in the final step of the solid-phase synthesis by mild treatment with trifluoroacetic acid (hereinafter abbreviated as "TFA"). In the solid-phase synthesis by this method, UV absorption spectra makes it possible to monitor the progress of reactions, i.e., the elimination of protecting groups for .alpha.-nitrogen in amino acids on a polymer support. This Fmoc-based strategy has made the synthesis of methionine- or cystein-containing polypeptides easier than before; as it has the synthesis of acid-sensitive tryptophan-containing polypeptides.
However, the procedure for the preparation of polypeptide amide by Fmoc strategy seems not to be well established, since in this approach, it is necessary to explore precursors of the amide function, preferably more acid-labile precursors than those hitherto employed in the Merrifield's solid-phase synthesis. At present, several resins coupled to benzylamine or benzhydrylamine have been introduced for this purpose. However, in order to prepare these resins, difficult-to-handle chloromethylated polystyrene resin has to be manipulated. In comparison with such laborious manipulation, the use of a handle-reagent, which can be directly introduced onto the commercially available polystyrene resin, is attractive from a practical viewpoint. As such a candidate, Albericio and Barnary introduced a modified benzylamine reagent, 5-[(2' or 4')-Fmoc-aminomethyl-3', 5'-dimethoxy] phenoxyvaleric acid (Albericio, F., Barnary, G.: Int. J. Peptide Protein Res., 30, 206 (1987))
However, this reagent involves two problems: one is that the material can be obtained through seven steps from the starting material, and the other is that the overall yield of the product is only 15%.
The inventors of the present invention conducted intensive studies in order to solve the above-described problems and found the following: by introducing a lower alkoxy group to both benzene rings in benzhydrylamine, the C--N linkage in benzhydrylamine is rendered acid-labile; by attaching a certain group such as a propionic acid group to benzhydrylamine, the latter could be effectively coupled to an aminomethylated polystyrene resin. The present invention has been accomplished on the basis of these observations.
The present invention provides novel benzhydrylamine derivatives of the following general formulas (I) and (II) which are useful as reaction reagents that are capable of efficient solid-phase synthesis of polypeptide amides. The present invention also provides a novel benzophenone derivative as an intermediate of these derivatives which is represented by the following general formula (III): ##STR2## (where R.sub.1 and R.sub.2 each independently represents a C.sub.1-3 lower alkyl; n is an integer of 1-4; l is 1 or 2, and m is 1 or 2); ##STR3## (where R.sub.1, R.sub.2, n, l and m have the same meanings as defined above; and Fmoc is a 9-fluorenylmethyloxycarbonyl group); and ##STR4## (where R.sub.1, R.sub.2, n, l and m have the same meanings as defined above).
Compounds of the general formula (I), (II) and (III) may be prepared by the following sequence of reactions, for example, starting with methyl 3-(4-methoxyphenyl)propionate:
1) Friedel-Crafts' condensation of methyl 3-(4-methoxyphenyl)propionate and p-methoxybenzoyl chloride;
2) Saponification of the resulting methyl ester;
3) Treatment with hydroxylamine (production of oxime);
4) Reduction of the resulting oxime with Zn (in acetic acid); and
5) Attachment of Fmoc to the resulting amino compound by treatment with Fmoc-OSu (Su stands for N-hydroxysuccinimidyl).
The resulting Fmoc reagent is loaded on an aminomethylated polystyrene resin by condensation with dicyclohexylcarbodiimide (hereinafter abbreviated as "DCC") in the presence of 1-hydroxybenzotriazole (hereinafter abbreviated as "HOBt"). This reaction is continued until the resin becomes negative to a Kaiser test. Before using the resin as a reaction agent, the Fmoc group attached is removed by treatment with dimethylformamide (hereinafter abbreviated as "DMF") containing 20% piperidine. Thus, 2,4'-dimethoxybenzhydrylamine resin anchored through the propionyl linkage (hereinafter named "Resin A") is readily prepared.
In addition, the Fmoc reagent is condensed onto an amino acid, for example, Ile which is bound to a PAM resin (PAM is the acronym for 2-phenylacetamidemethyl), then the Fmoc group is removed as described above to produce another resin for Fmoc-based solid-phase synthesis (hereinafter named "Resin B"). In this resin, the amino acid (e.g. Ile) used serves to monitor the amount in which the polypeptide amide is liberated by acid hydrolysis.
Methods of synthesizing the compounds of the present invention, as well as Resins A and B which are reaction reagents containing those compounds are described hereinafter in detail with reference to working examples. In order to identify the respective compounds, chromatography, NMR, MS and IR analyses were conducted with the following apparatus: Chromatography: Using a thin layer of silica gel (Kiesel-gel G, Merck), chromatography was conducted [Rf.sub.1, CHCl.sub.3 ; Rf.sub.2, CHCl.sub.3 -MeOH-H.sub.2 O (8:3:1)].
Nuclear magnetic resonance (.sup.1 H-NMR) spectra: Taken on a JEOL FX-200 spectrometer, with tetramethylsilane used as an internal standard.
Mass spectra (MS): Taken on a JEOL JMS-01SG-2 spectrometer.
Infrared (IR) absorption: Measured with a Hitachi 215 spectrophotmeter.